Targeting RNA modifications with pharmacological agents: New frontiers in cancer therapy

Abstract The N6‐methyladenosine (m6A) RNA modification has gained significant prominence as a new layer of regulatory mechanism that governs gene expression. Over the past decade, various m6A regulators responsible for introducing, eliminating, and recognising RNA methylation have been identified. Notably, these m6A regulators often exhibit altered expression patterns in cancer, occasionally offering prognostic value. Nonetheless, the complex roles of these regulators in human cancer pathology remain enigmatic, with conflicting outcomes reported in different studies.In recent years, a multitude of inhibitors and activators targeting m6A regulators have been reported. Several of these compounds have demonstrated promising efficacy in both in vitro and in vivo cancer models. These findings collectively underscore the dynamic landscape of m6A regulation in cancer biology, revealing its potential as a therapeutic target and prognostic indicator.

quantitative strategies and knowledge concerning proteins that regulate m6A. 5 The catalytic component of the m6A writer, Methyltransferase-Like Protein 3 (METTL3), was identified in 1997, 25 years after the first discovery of m6A modification in 1974. 6,7ethyltransferase-Like Protein 14 (METTL14) was then reported as the second protein in the m6A writer complex, functioning together with METTL3 to enhance its activity. 8Completing the core m6A-methyltransferase complex, Wilms's tumour 1-associating protein (WTAP) interacts with the METTL3/METTL14 to allow their localisation into nuclear speckles. 91][12] VIRMA mediates mRNA m6A modification in 3'UTR and near stop codon, explaining the enrichment of m6A modification at these specific regions of mRNAs. 11,13ZC3H13 interacts with WTAP, bridging the METTL3/METTL14/WTAP complex to the other cofactors, which is also essential for localising the complex. 12,14HAKAI is essential for the stabilisation of core components of the complex where disruption of HAKAI leads to degradation of VIRMA and ZC3H13. 15,16RBM15 and RBM15B have redundant functions in interacting with WTAP, responsible for guiding the complex to the specific RNA target, XIST. 10 They bind U-rich regions of the RNA, 10 but whether they are essential for all m6A modification by the METTL3/ METTL14 complex remains elusive.
Other m6A writers described act on RNA species other than mRNAs.METTL16 installs m6A on U6 snRNA and S-adenosylmethionine (SAM) synthetase pre-mRNA. 17hile various ncRNAs, lncRNA, and pre-mRNA were reported to associate with METTL16, whether all these interactions involved m6A methylation demand further research. 18][21] Two established m6A erasers are Fat mass and obesity-associated protein (FTO) and alkB homologue 5 RNA demethylase (ALKBH5).FTO is associated with human obesity and energy homeostasis, demethylating m6A in cellular mRNA and other RNA species. 22,23LKBH5, on the other hand, specifically demethylates m6A-marked mRNA or sometimes m6A-marked ssDNA, showing minimal activity towards m6A rRNA or other types of RNA modifications. 24The substrate specificity of FTO and ALKBH5 is influenced by the conformational diversity of RNA, determined by both the sequence and the conformational changes due to m6A modification. 25he functional outcome of an m6A modification is predominantly determined by the m6A-binding proteins that 'read' the modification, including the YTH domaincontaining proteins (YTHDC1-2), the YTH domain family (YTHDF1-3), the insulin-like growth factor 2 mRNA binding proteins (IGF2BP1-3), and the HNRNP family (HNRNPA2B1, HNRNPC, HNRNPG).
Nuclear m6A readers regulate alternative splicing and nuclear-cytoplasmic export of RNA.YTHDC1 selectively targets GG(m6A)CU over GA(m6A)CU 26 and recruits the splicing factor SRSF3 to promote exon inclusion.YTHDC1 also promotes nuclear-cytoplasmic export by recruiting NXF1. 27Other nuclear readers involved in pre-RNA processing are HNRNPC and HNRNPG. 28,29However, the mechanism of regulation and how they select their targets remain elusive.HNRNPA2B1 binds m6A-marked primary microRNAs and promotes miRNA processing and exosome sorting. 30Interestingly, HNRNPA2B1 has a greater affinity to non-methylated RNA than methylated RNA, suggesting a potential 'm6A-switch' mechanism for regulating RNA metabolism rather than an 'm6A-promoting' mechanism. 31ytoplasmic m6A readers regulate RNA stability and translation.3][34] YTHDC2 also promotes RNA degradation. 35In contrast, IGF2BPs promote RNA stability and translation. 36YTHDF1 and YTHDF3 enhance mRNA translation by interacting with translation initiation factors, including eIF3, eIF4A3, and eIF4A3. 37,38owever, neither simultaneous nor independent knockout of YTHDF1-3 reduces the translation efficiency, suggesting that they are not regulating translation in cells at homeostasis. 34While the role of YTHDFs in mRNA translation remains controversial, it appears that they may be associated with stress granule formation and possibly regulate m6A-associated translation of a limited number of mRNAs under stressful conditions in physiological and pathological conditions, including cancer. 39,40 2 |THE COMPLEX ROLE OF m6A

REGULATORS AS ONCOPROTEINS AND TUMOUR SUPPRESSORS
Given their key roles in normal physiology, m6A writers, erasers, and readers have been implicated in diverse human cancers.Notably, the m6A regulators often display opposing roles as oncoproteins and tumour suppressors (Tables 1-3).In this section, we will discuss the complex role of some m6A regulators in tumorigenesis, leading us to explore the prospects of potential therapeutic approaches in the following section.

METTL3 and METTL14
METTL3 is overexpressed in Acute Myeloid Leukaemia (AML) cells compared to healthy haematopoietic cells.Promoter-bound METTL3 promotes the translation of oncoproteins, including SP1, facilitating AML development. 41METTL3's tumorigenic effect involves widespread mRNA targets, including c-MYC, BCL2, PTEN, and MDM2. 42,43Similarly, METTL14 is significantly upregulated in AML carrying t(11q23), t (15;17), or t (8;21). 44echanistically, METTL14 increases m6A levels on the MYB and MYC transcripts, preventing cell differentiation but enhancing survival and proliferation. 44onversely, METTL3 and METTL14 act as tumour suppressors in Triple-Negative Breast Cancer (TNBC), where their downregulation leads to tumour growth and metastasis.Mechanistically, METTL3 is repressed by miR-34c-3p in TNBC, 45 and it negatively regulates the expression of the oncogenic COL3A1. 46METTL3 depletion also contributes to tumour progression in Hormone Receptor Positive (HR+) and Human Epidermal Growth Factor Receptor 2 Negative (HER2-) breast cancer. 47However, the roles of METTL14 and METTL3 in breast cancer, in general, are controversial.Despite the tumour-suppressive functions evidenced in the experimental studies on TNBC and HR + HER2-breast cancer, other studies have reported the oncogenic roles.9][50][51][52] These studies covered a wide range of breast cancer subtypes including TNBC, HER2+, and HR+ breast cancer.METTL14 was also reported to promote breast cancer cell proliferation, migration and invasion by methylating miRNAs. 53,54The complex role of the methyltransferase complex in breast cancer indicates a need for further exploration of how m6A affects carcinogenesis in subtype-specific breast cancer.
Overexpression of METTL3 in glioblastoma stem cells (GSCs) has been correlated with a poor prognosis for glioblastoma and its silencing in GSCs has been shown to reduce tumour growth in vivo. 556][57][58] Mechanistically, METTL3 stabilises DNA repair genes MGMT and APNG, thereby enhancing sensitivity to chemotherapy with TMZ. 58Additionally, it stabilises SOX2, facilitating SOX2-dependent DNA repair during radiotherapy.In colorectal cancer (CRC), upregulated METTL3 is associated with a poor prognosis and promotes cellular proliferation and metastasis in vitro and in vivo.Examples of downstream RNA affected are CCNE1 which regulates the cell cycle, 59 HK2 and SLC2A1 which are involved in glycolysis, 60,61 and SOX2 and MYC which interact with key proliferative pathways such as EGFR, Akt, NOTCH and Wnt signalling. 62,63Notably, METTL3 also decreases the stability or translation efficiency of tumour-suppressor genes.][66] METTL3 also targets ncRNAs, including pri-miRNA, to regulate their processing, leading to aberrant expression of their cognate target oncogenes and tumour-suppressor genes. 67,680][71] The contrasting effects of METTL3 and METTL14 on CRC progression despite their complex formation and catalytic enhancement may be attributed to their preference for different targets, leading to diverse downstream pathways. 8,71Moreover, the tumoursuppressive role of METTL14 in p53-wild-type CRC cells, while not significantly affecting p53-mutant or p53-null CRC cells, highlights the influence of tumour heterogeneity on m6A regulators' roles. 72The observed controversies can be attributed, at least partially, to this heterogeneity.More examples of the complex roles of METTL3/METTL14 in diverse cancers are shown in Table 1.

| Other m6A writers
The recently identified m6A writers, METTL5 and METTL16, have also been implicated in cancer (Table 1).METTL5 is overexpressed in breast cancer, 73 pancreatic cancer, 74 uterine corpus endometrial carcinoma, 75 and hepatocellular carcinoma (HCC), 76 but significantly decreased in gastric cancer tissues compared to adjacent normal tissues and intestinal metaplasia tissues. 77METTL16 facilitates the progression of breast, 78 gastric, 79 lung, 80 AML, 81 and liver cancers. 82Conversely, METTL6 expression is positively correlated with the overall survival of endocrine system tumours. 83

| m6A erasers: FTO and ALKBH5
The role of FTO in breast cancer is complex and contradictory.One on hand, FTO promotes breast cancer cell proliferation, colony formation, cellular invasion, and metastasis in vitro and in vivo. 84 mRNA and induces its degradation to inhibit apoptosis while increasing cell proliferation. 85Demethylation of m6A at miR-181p-3p by FTO inhibits the miRNA function to allow expression of the oncogenic ARL5B, promoting cellular invasion and migration. 86In this context, FTO inhibition could be a potential therapeutic strategy for breast cancer.Conflicting with the above, FTO downregulation was also reported in breast cancer, promoting tumour progression and metastasis via enhancing expression of mesenchymal markers including SNAI2, VIM, FN1, NT5E, SNAI1, MMP2 and ZEB1 while decreasing epithelial markers FSTL3, KRT18 and TJP1. 87Moreover, FTO-depleted cells showed increased Wnt signalling and are sensitive to Wnt inhibitor therapy. 879][90] In these cases, FTO overexpression leads to the downregulation of ASB2 and RARA proteins, promoting the overexpression of oncogenic MLL and the activation of the PDFGRB/ERK pathway. 89The presence of other markers, such as NPM1 mutation type A, would induce FTO expression, resulting in TP53INP2 upregulation which promotes autophagy and leukaemia cell survival. 88,90Such mechanisms suggest potential correlations between m6A regulators and specific AML subtypes, highlighting the potential for precision treatments targeting m6A modifications in AML.
FTO is downregulated in ovarian cancer stem cells and tumours. 91Downregulation of FTO increases the m6A level in the SNAI1 transcript, enhancing its stability via an IGF2BPs-dependent manner and promoting epithelialto-mesenchymal transition. 92FTO inhibits ovarian cancer stem cell self-renewal by upregulating PDE1C and PDE4B, which subsequently block the cAMP signalling pathway. 91owever, FTO was also reported to be upregulated in ovarian tumour tissues, increasing cellular viability and autophagy function but decreasing apoptosis. 93Therefore, the role of FTO in ovarian cancer remains controversial, possibly due to the different cancer models.The mechanism of action of FTO in ovarian cancer demands further research.
Similarly, oncogenic and tumour-suppressive roles of ALKBH5 have been reported.ALKBH5 enhances the expression of FOXM1 via demethylation, promoting stem-like cell proliferation and tumourigenesis in glioblastoma. 946][97] In contrast, ALKBH5 functions as a tumour suppressor in thyroid cancer and Non-Small Cell Lung Cancer (NSCLC) by reducing the expression of TIAM1 and YAP, respectively. 98,99In CRC, downregulation of ALKBH5 is associated with poor prognosis. 100,101Downstream transcripts were identified to be PHF20, FOXO3, and SLC7A11, in which the stability of PHF20 and SLC7A11 are decreased by ALKBH5 while the FOXO3 mRNA's stability is enhanced. 100,102,103Recently reported examples of FTO and ALKBH5-mediated cancer development and progression are detailed in Table 2.

| m6A readers
The role of m6A readers in cancer is also complex (Table 3).For example, the overexpression of all YTHDF1-3 has been implicated in breast cancer progression and metastasis.In breast cancer, HIF1α induced by hypoxia inhibits miR-16-5p, which under normal conditions targets and inhibits YTHDF1 via mRNA 3′UTR. 104However, hypoxia-induced YTHDF1 overexpression enhances the translation of PKM2 and subsequently upregulates glycolysis. 104YTHDF1 also upregulates the translation of oncogenic FOXM1. 105THDF3, a prognostic biomarker for breast cancer, promotes brain metastasis by enhancing the expression of key metastatic genes including GJA1, ST6GALNAC5, and EGF. 106,107YTHDF2 mediates the m6A-dependent degradation of the lncRNA FGF14-AS2, and patients with high YTHDF2 and low FGF14-AS2 expression have worse distant metastasis-free survival. 108n HCC, YTHDF1 is upregulated by HIF1α under hypoxic conditions, facilitating the translation of autophagy-related genes ATG2A and ATG14 in an m6Adependent manner. 109YTHDF1 also positively regulates ANLN, promoting HCC bone metastasis. 110YTHDF3 is overexpressed in HCC and correlates with poor prognosis. 111While YTHDF3 is generally accepted to promote RNA degradation or enhance translation, it was found to stabilise PFKL mRNA, leading to increased expression and promoting aerobic glycolysis and carcinogenesis. 111In contrast, YTHDF2 is downregulated under hypoxic conditions, 112 and forced YTHDF2 expression promotes the degradation of oncogenic EGFR mRNA, suppressing HCC cell proliferation and growth in vitro and in vivo. 112However, contrary to that study, other research has demonstrated that YTHDF2 is a negative downstream target of a frequently downregulated miRNA in HCC, miR-145. 113Recently reported examples of m6A reader-mediated cancer development and progression are detailed in Table 3.

PLAYERS IN CHEMORESISTANCE
Chemoresistance remains a life-threatening obstacle in cancer biology and clinical practice.Multiple factors and mechanisms have been identified, carrying important clinical implications.m6A regulators were linked to chemoresistance (Table 4), providing a potential combination therapeutic strategy.
Both upregulation and downregulation of METTL3 impact cancer sensitivity to chemotherapy, further highlighting the intricate role of m6A regulators.5][116] Downregulation of METTL3 has been reported in HR + HER2-breast cancer, promoting resistance to doxorubicin, paclitaxel, and cisplatin. 47These seemingly contradictory findings may arise from different downstream readers recognising the m6A-marked mRNA.Despite similarities in DNA-damaging chemotherapies' primary mode of action, each therapy targets multiple pathways providing additional effects and interactions with cells and the tumour microenvironment (TME).For instance, doxorubicin induces immunogenic cell death, stimulating immune responses, and inhibiting regulatory T cells. 117Thus, its anti-tumour effect extends beyond DNA damage to immunomodulation.The complexity and the heterogeneity of chemotherapeutic response in different cancer subtypes contribute to these conflicting results.
9][120][121][122] Downregulation of METTL3 also associates with resistance to daunorubicin and cytarabine in AML. 114he m6A erasers FTO and ALKBH5 also mediate chemoresistance in cancer.The upstream regulator STAT3 promotes FTO expression in breast cancer, resulting in doxorubicin resistance that can be reversed by FTO knockdown. 123FTO overexpression targets apoptosisinducing factor SIVA1, conferring 5-FU-resistance in CRC cells. 124Consistently, inhibition of FTO pharmacologically or genetically reduced the 5-FU tolerance of CRC xenograft models. 124These results suggest that FTO inhibitors hold the potential for overcoming chemoresistance, which is discussed further in the below section.However, FTO also exhibits a protective role, its downregulation was found in platinum (Pt)-resistant ovarian cancer cells and forced expression increases sensitivity to Pt in vitro and in vivo. 124LKBH5 mediates Temozolomide resistance in glioblastoma by demethylating the SOX2 transcript, increasing its expression. 125In breast cancer, ALKBH5 demethylates GLUT4 mRNA, enhancing its stability and correlating with resistance to trastuzumab and lapatinib. 126In addition, ALKBH5 targets the WIF-1 transcript, enhancing its expression and activating Wnt signalling, resulting in gemcitabine resistance in adenocarcinoma. 127THDF1 overexpressed in cisplatin-resistant CRC cells, promotes GLS1 protein expression, elevating glutamine metabolism and cisplatin resistance. 128YTHDF1 knockdown enhances sensitivity to Adriamycin, cisplatin, and Olaparib in breast cancer cells. 129YTHDF2 is also involved in cisplatin resistance, the key downstream targets were found to be AXIN1 in cervical cancer, and CDKN1B in intrahepatic cholangiocarcinoma. 130,131YTHDF3 is highly expressed in oxaliplatin-resistant CRC tissue, facilitating eIF2AK2 and eIF3A recruitment on mRNAs to regulate translation. 132In contrast, YTHDC1 is downregulated in clear cell renal cell carcinoma (ccRCC) and reduces sensitivity to sunitinib. 133Apart from chemoresistance, YTHDF3 and YTHDC2 correlate with radiotherapy resistance in cervical cancer and nasopharyngeal carcinoma.Mechanistically, YTHDF3 promotes RAD51D translation and YTHDC2 activates the IGF1R/ATK/S6 signalling axis, both in an m6A-dependent manner. 134,135he IGF2BPs also mediate chemoresistance.Overexpression of IGF2BP1 mediates doxorubicin resistance via stabilising the mRNA of oestrogen-related receptor alpha (ERRα) and ABCB1. 136,137Similarly, IGF2BP2 overexpression causes chemoresistance to cytarabine, dexamethasone, vincristine, and venetoclax in T-cell acute lymphoblastic leukaemia (T-ALL) by recognising m6A-marked NOTCH1 mRNA and stabilising it. 138

| m6A REGULATORS ARE KEY PLAYERS IN CANCER IMMUNOLOGY
Cancer immunotherapy has revolutionised the cancer treatment in the last decade, with notable successes such as immune checkpoint blockades (ICBs) and CAR-T cell therapy. 139The influence of m6A regulators extends beyond cancer cells to encompass immune cells within the TME, potentially influencing the outcomes of immunotherapies.Consequently, m6A regulators emerge as promising targets for combination therapy with ICBs or cell therapies, as detailed in Table 5.
METTL3 suppresses anti-tumour immune response by reducing granzyme B and interferon gamma-positive CD8+ T cell infiltration. 140METTL3 depletion synergises with anti-PD-1 blockade, impeding tumour progression in various in vivo models, including CRC, melanoma, and HCC. 140,141Recently, in vivo models demonstrated that METTL3 inhibition is equally efficacious to anti-PD-1 therapy and combination of both provide synergism.Nasopharyngeal carcinoma (NPC).Mechanistically, catalytic inhibition of METTL3 results in dsRNA formation and potent cell-intrinsic interferon responses that can stimulate anti-tumour immunity, which is distinct to the mechanism of the current ICBs and cell therapy.Importantly, the combination of anti-PD1 and METTL3 inhibitor can augment antitumor immunity to eliminate malignant clones insensitive to these agents alone, suggesting that METTL3 and ICBs work through distinct but complementary pathways. 142owever, conflicting findings suggest that selective ablation of METTL3 in myeloid cells remodels the TME, increasing M1/M2-like tumour-associated macrophage and regulatory T (Treg) cell infiltration. 143Moreover, myeloid-specific METTL3 depletion attenuates efficacy of anti-PD-1 in melanoma.This level of contradictory might attribute to the different functions of METTL3 in cancer and immune cells, underscoring the complexity of targeting METTL3 in cancer immunotherapy.
FTO-mediated m6A demethylation, on the other hand, elevates the expression of transcription factors c-Jun, JunB, and C/EBPβ, thereby enhancing glycolytic metabolism and inhibiting CD8+ T cell infiltration. 144Others immune related genes upregulated by FTO-mediated demethylation includes PD-1, CXCR4, and SOX10. 145Preclinical models of melanoma and CRC reveal synergism between FTO inhibition and anti-PD-1 therapy. 144,145The m6A eraser ALKBH5, when deleted, sensitises tumours to ICBs in vivo. 146Mechanistically, ALKBH5 positively regulates Mct4/Slc16a3 and lactate levels during anti-PD-1/GVAX treatment, increasing Treg cells and myeloid-derived suppressor cells (MDSCs) accumulation in TME.Moreover, lower ALKBH5 expression in melanoma correlates with better response to anti-PD-1 therapies such as pembrolizumab or nivolumab.However, the paradoxical role of ALKBH5 in different cancers is evident, as it positively regulates PD-L1 expression in intrahepatic cholangiocarcinoma. 147Patients with strong nuclear expression patterns of ALKBH5 exhibit greater sensitivity to anti-PD-1 therapy, emphasising the diverse functions of m6A regulators across cancer types.
Similarly, the roles of m6A readers vary substantially in different cancer types.In CRC, YTHDF1 impairs antitumour immunity by negatively regulating CD8+ T cell infiltration while upregulating CXCL1 to promote MDSCs infiltration. 148Consistently, YTHDF1 knockout increases anti-PD1 efficacy and CD8+ infiltration in CRC. 148,149owever, almost all subsets of tumour-infiltrating lymphocytes including CD8+ T cells are high in high YTHDF1 and YTHDF2 lung cancers, suggesting distinct downstream target genes of m6A readers between cancer types. 150Further examples of m6A readers influencing antitumor immunity are detailed in Table 5.
It is crucial to note that genetic knockout or siRNAmediated depletion may differ from pharmacological inhibition, which holds greater relevance in clinical applications.The subsequent section will explore the effects of inhibiting m6A regulators with small molecules in combination with immunotherapy.

| THERAPEUTIC POTENTIAL
While the relationship between m6A and cancer has been extensively studied, the development of therapeutics targeting m6A regulators is still in its infancy.

| M6A writer-METTL3
The study of m6A modulators, including METTL3 inhibitors, has gained increasing attention due to their roles in regulating gene expression in cancer cells.Targeting METTL3 based on its diverse functions holds promise for developing precision cancer therapies (Table 6).

| Competitive inhibitors
METTL3 was extensively reported as an oncoprotein (Table 1); therefore, METTL3 inhibitors have the potential to be anti-tumour drugs.The first reported METTL3 inhibitor, adenosine, competitively binds to the SAM binding site as METTL3 is an S-adenosyl-L-methioninedependent methyltransferase. 151Subsequent docking studies of 4000 adenosine-moiety-containing compounds into the SAM binding site identified 70 hits. 151Experimental validation of these hits led to the discovery of 7 candidates with promising inhibitory effects.However, the anti-tumour efficacy of these adenosine derivatives was not tested in cancer cell lines or mouse models.Furthermore, the selectivity of this class of inhibitors remains to be examined.
A structure-based drug discovery approach led to the discovery of the potent and selective UZH1a. 152Co-crystal of UZH1a-METTL3 revealed a significant conformational rearrangement (6Å displacement) of the Lys513 side chain, distinguishing it from the Lys513 orientation observed in the co-crystal structure of METTL3 with sinefungin, a non-selective inhibitor of SAM-dependent methyltransferases.This unique conformation of METTL3 induced by UZH1a is believed to contribute to its selectivity.Notably, UZH1a possesses favourable physicochemical properties, such as low molecular weight and good cellular permeability.Demonstrating high-nanomolar potency in a T A B L E 6 Therapies targeting METTL3.

Anti-tumour effect (In vitro)
Anti-tumour effect (In vivo)

Cpb-564
Renal injury and inflammation [159]   STM2457 Human AML cell lines treated with STM2457 shows significant growth reduction in a concentrationdependent manner.
Treatment leads to impairment of engraftment and AML expansion in vivo and significantly prolongs the mouse lifespan.
AML [153]   Allograft rejection [276]   The IC50 values of cisplatin and etoposide are significantly decreased after treatment with STM2457.Significantly inhibits the growth rate of the xenografts when combined with chemotherapy.
Liver cancer, pancreatic cancer [155]   Allosteric inhibitor CDIBA Shows dose-dependent antiproliferative activities in multiple AML cell lines.
AML [160]   Eltrombopag Shows anti-proliferative in multiple AML cell lines, and demonstrates synergistic effect when combined with venetoclax and cytarabine.
AML [161]   Activator Piperidine derivative and piperazine derivative compounds Shows proliferative effect and increases m6A level in HEK293 cells.[162]  biochemical assay, UZH1a effectively reduces the m6A/A ratio in the AML MOLM-13 cell line and the osteosarcoma U1OS cell line.However, the anti-tumour effect of UZH1a has yet to be tested in vitro or in vivo.Through high-throughput screening (HTS) of 250,000 compounds, STM2457 was identified as a potent (IC50 = 16.9 nM) and selective METTL3 inhibitor. 153STM2457 competitively binds the SAM binding pocket.Consistent with the oncogenic role of METTL3 in AML, [41][42][43] treatment of mouse and human AML cells with STM2457 consistently demonstrates growth reduction, myeloid differentiation, and cell cycle arrest. 153Moreover, treatment with STM2457 induces apoptosis in human and mouse AML cell models but not in normal non-leukaemic haematopoietic cells, which could be advantageous in minimising side effects.The result is replicable in in vivo model, expanding the lifespan of mice with minimal toxicity observed. 153In addition to the oncogenic role, METTL3 is responsible for SCLC chemoresistance and STM2457 successfully reversed the chemoresistant in vitro and in vivo. 121ery recently, the competitive inhibitor STM3006 was published. 142It has 20-fold increased cellular potency compared with STM2457 and potently inhibits proliferation but induces apoptosis of multiple cell lines.While STM3006 is structurally distinct from STM2457, they have very similar binding poses revealed by x-ray crystallography, possibly explaining the high selectivity of both inhibitors.In addition, STM3006 inhibition results in a cell-intrinsic interferon response and enhanced antigen-dependent tumour killing by cytotoxic CD8+ T cells.STM3006 has rapid metabolism and has no efficacy in vivo but its improved oral availability version, STC-15, is now under phase I clinical trial in solid cancer (NCT05584111).
Virtual screening of 1012 South African natural products led to the identification of three candidates, SANCDB0370, SANCDB0867, and SANCDB1033, derived from Buddleja salviifolia, Croton gratissimus, and Struthiola argentea, respectively. 154These candidates exhibit more negative free energy than STM2457.The in silico analysis suggested that these compounds possess drug-like properties and lower toxicity compared to STM2457. 154It is important to note that while the computational methods provided valuable insights into the candidates' properties, wet-lab experiments are yet to be conducted to validate their activity in vitro or in vivo.
Quercetin, a competitive METTL3 inhibitor with micromolar potency, was recently discovered through the virtual screening of natural products. 155Quercetin is cell permeable and capable of decreasing mRNA m6A levels in human pancreatic adenocarcinoma cells. 155Quercetin's anti-proliferative effects have been confirmed in various cancer cell lines, including liver, lung, breast, and pancreatic cancer cells. 155Notably, quercetin has been studied for its anti-tumour properties for over two decades, and clinical trials have shown no reported toxicity or side effects. 156,157However, quercetin is a non-specific inhibitor with pleiotropic effects and can target multiple enzymes, including DNA methyltransferases and histone deacetylases. 158Despite its potential safety as a cancer patient supplement, the lack of selectivity for METTL3 suggests the need for further optimization.Virtual screening also revealed Cpd-456, while it has demonstrated potential in protecting against renal injury and inflammation, its anti-tumour efficacy remains unstudied. 159

| Allosteric inhibitors
Besides competitive inhibitors, two allosteric inhibitors were found to inhibit METTL3.The first allosteric inhibitor, known as CDIBA, potently inhibits the METTL3/ METTL14 complex but not the individual METTL3 and METTL14 subunits, indicating its simultaneous binding to METTL3 and METTL14. 160In contrast, Eltrombopag exhibits similar inhibitory activity on the complex and METTL3 alone but not on METTL14. 161Computational studies consistently suggested that the putative binding site of Eltrombopag is on the METTL3. 161Additionally, Eltrombopag selectively targets METTL3 over other histone methyltransferases, including DOT1L, G9a, PRMT1, SETD2, and SMYD3.Both CDIBA and Eltrombopag demonstrated an anti-proliferative effect in the AML cell line MOLM-13, leading to a reduction in the m6A level. 160,161

| METTL3 activator
Four potential METTL3 activators were identified via virtual screening. 162The series of compounds containing piperidine and piperazine rings showed high docking efficiencies.These compounds, which partially occupy the SAM pocket, seem to activate the methylation activity of METTL3/METTL14.The ability of these compounds to reactivate METTL3 to suppress cancer subtypes associated with METTL3 downregulation remains to be explored.

| M6A eraser-FTO
FTO has long been studied as a promising molecular target for treating obesity. 163Therefore, more inhibitors have been developed for FTO than for METTL3 (Table 7).These FTO inhibitors are primarily competitive, binding Breast cancer [170]   CHTB Obesity [167]   N-CDPCB Obesity [165]   Meclofenamic acid 2 Exerts a substantial inhibitory effect on the growth and the self-renewal of various cancer cell lines.
PBT003-grafted mice treated with MA2 has smaller tumour size with decreased tumour luciferase activity and prolonged survival.
Glioblastoma [56,174]   MA Shows significant synergistic effects with gefinitib in gefinitib-resistant NSCLC cell lines.
13a Inhibits colony formation of NB4 cells in concentration-dependent manner.Improves the survival rate of MONOMAC6transplanted NSG mice.
AML [179]   FB23 and FB23-2 Suppresses proliferation and promotes the differentiation/apoptosis of human AML cells and primary blast AML cells.
Inhibits the progression of human AML cell lines and primary cells in xenotransplanted mice and shows prolonged survival.
AML [178]   R-2HG Exhibits a broad growth-suppressive activity in leukaemia in general.
Treatment leads to decrease of leukaemic blasts in mice with sensitive cells.
Leukaemia and glioma [183,184]   Glycolysis is suppressed by R-2HG in sensitive leukaemia cells.

Mode of action Therapy Anti-tumour effect (In vitro)
Anti-tumour effect (In vivo)

GNPIPP12MA nanoparticles
The combination of GNPIPP12MA with PD-L1 blockade significantly inhibits leukaemia progression and metastasis in a mouse model. [180]

Dac51
Enhances release of cytokines and cytotoxic capacity in T cells cocultured with Dac51-pretreated B16OVA tumour cells.
Glioblastoma Multiforme [193]   Uncompetitive inhibitor to either the substrate (i.e.methylated ssRNA/DNA) or the cofactor (i.e.2-oxoglutarate (2OG)) binding site.To the best of our knowledge, no allosteric inhibitor has been reported.

| Competitive inhibitors
Via virtual screening, rhein and N-CDPCB were identified as competitive FTO inhibitors, increasing the m6A level in cells. 164,165Molecular modelling revealed that rhein binds to m3T, 2OG, and Fe2+ binding sites, disrupting the cofactor and substrate binding. 166Similarly, N-CDPCB binds to the substrate-binding site by occupying the space between an antiparallel sheet and the extended C-terminal of the long loop of FTO. 165Given that the loop of FTO is not conserved in other mammalian ALKB members, N-CDPCB is likely to be a selective inhibitor. 165Another competitive inhibitor, CHTB, also binds to the non-conserved site of FTO, suggesting good selectivity. 167In the absence of experimental validation, the selectivity profile of these competitive inhibitors remains largely unknown.Furthermore, the anti-tumour efficacy of N-CDPCB and CHTB has not been tested.More FTO inhibitors were discovered through structure-based virtual screening, encompassing entacapone from FDA-approved drugs, two quinolone derivatives from the ZINC library, and 18,077 and 18,079 from the commercial database Specs (https:// www.specs.9][170] Entacapone, an FDA-approved therapy for Parkinson's disease in combination with levodopa and carbidopa was found to induce apoptosis in oesophageal cancer cell lines YM-1 and KYSE-30. 171,172The two quinolone derivatives, identified as FTO inhibitors, were originally investigated for supporting the survival of dopamine neurons in neurodegenerative disease and their anti-tumour efficacy requires further studies. 169In vitro studies demonstrated that 18,097 significantly suppresses the colony number of cancer cells. 170Moreover, 18,097 enhances the sensitivity of HeLa cervical cancer cells and MDA-MB-231 breast cancer cells to cisplatin and doxorubicin.It also inhibits cancer cell invasion by downregulating the expression of matrix metalloproteinase 2 (MMP2), fibronectin (FN), and vimentin. 170eclofenamic acid (MA) is a nonsteroidal antiinflammatory drug approved by the FDA in 1980, commonly used to treat pain and inflammation associated with osteoarthritis, rheumatoid arthritis, and menstrual cramps. 173In 2015, it was found to selectively inhibit FTO's demethylation activity compared to other ALKB Family members. 174A prodrug called MA2 has also been developed, featuring an extra ethyl ester group to increase cell penetration.Upon hydrolysis of the ester, MA2 yields MA within cells.Both MA and MA2 have been studied in cancer models.In mice engrafted with glioblastoma stem cells (GSCs), MA2 treatment significantly reduced tumour size, and prolonged survival, suggesting the therapeutic potential of increasing m6A level through FTO inhibition. 174Additionally, MA2 and the previously introduced Rhein were shown to restore nilotinib sensitivity in tyrosine kinase inhibitor (TKI) resistant leukaemia in vitro and in vivo. 175MA also reverses Gefitinib resistance in NSCLC cells, showing synergistic effects with Gefitinib in Gefitinib-resistant NSCLC cells. 176Nevertheless, others have reported the tumour-suppressive role of FTO, 177 emphasising the need for further research before considering clinical studies involving FTO inhibitors in cancer.
Several competitive FTO inhibitors have been developed by optimising MA to enhance potency, target selectivity, and pharmacokinetics.Among all synthesised analogues, FB23 stand out with the highest potency, showing an approximately 140-fold increase over MA. 178 The derivative of FB23, named FB23-2, demonstrates improved cellular permeability, leading to increased m6A levels and exhibits anti-proliferative efficacy in various AML cell lines. 178Moreover, FB23-2 inhibits AML progression in xeno-transplanted mice, resulting in prolonged survival. 178The selectivity of MA is retained in these optimised inhibitors by preserving the benzyl carboxylic acid that interacts with the non-conserved loop in FTO. 174,178ubsequent optimisation of FB23 led to the discovery of Dac51 which forms additional hydrogen bonds with the FTO protein, improving potency. 144Considering FTO's role in the TME remodelling and its involvement in immune surveillance, co-culturing Dac51-pretreated B16OVA melanoma cells with T cells demonstrated enhanced cytokines release and elevated cytotoxic capacity. 144In vivo treatment with Dac51 increased the proportion of infiltrated CD8+ T cells in the TME and effectively inhibited tumour growth.Furthermore, combining Dac51 with an immune checkpoint blockade significantly prolonged the survival of mice compared to monotherapy. 144he design and synthesis of FB23 analogues, along with Structure-Activity Studies led to the discovery of compound 13a.It significantly inhibits FTO demethylation in vitro, suppresses AML cell proliferation, and improves the survival of MONOMAC6-grafted mice without displaying apparent off-target effects. 179ao et al. enhanced the efficacy of MA in tumour cells using nanoparticle technology for targeted delivery. 180hey developed GNCP12, a nanocluster with a 12-mer peptide (GGGCDLRSAAVC), which specifically targets C-type lectin-like molecule-1 that overexpressed on AML cells and CD34 + CD38+ leukaemic stem cells (LSCs).By incorporating GSH-S-DNP, a GSH derivative, as the imprinting template to create a hydrophobic pocket in the nanoparticle, GNCP12 binds the thiol group of GSH via ligand exchange in the hypoxic bone marrow.This triggers the selective release of loaded MA in the presence of GSH, enabling the targeted killing of AML cells and LSCs.Combining this nanoparticle therapy, termed GNPIPP12MA, with PD-L1 blockade effectively inhibited leukaemia progression and metastasis in the preclinical mouse model. 180uff et al. employed the binding pocket occupied by the selective MA to rationally design unique inhibitors while maintaining selectivity. 181They identified the pyrimidine scaffold as a promising replacement for the benzyl carboxylic group of MA, which provides the necessary selectivity.Subsequently, fragment growth was directed towards the unoccupied binding pocket, leading to the discovery of FTO-4.FTO-4 increases the m6A level of GSCs and impairs self-renewal in GSC-derived neurospheres. 181Further optimisation of FTO-4 led to FTO-43, which exhibits antiproliferative efficacy in multiple in vitro cancer models, including AML, glioblastoma, and gastric cancer. 182-2-hydroxyglutarate (R-2HG), a metabolite produced by mutant isocitrate dehydrogenases (IDHs), has been shown to inhibit FTO demethylation activity, leading to the downregulation of the oncogenic MYC.183 In xenografted mice experiments, both direct injection of R-2HG and IDH1R132H-mediated R-2HG generation significantly inhibited AML progression, indicating therapeutic potential.Additionally, Qing et al. showed that R-2HG inhibits glycolysis in AML by suppressing FTO's activity.184 This understanding sheds light on how R-2HG may contribute to resistance to mutant IDH inhibitors.Consequently, combining a mutant IDH inhibitor with an FTO inhibitor like R-2HG may hold therapeutic potential in treating resistant AML.
Saikosaponin-D (SsD), a naturally occurring triterpenoid saponin found in the roots of Bupleurum falcatum, competitively inhibits the demethylation activity of FTO. 185Like MA, SsD has shown the ability to overcome FTO/m6A-mediated leukaemia resistance to TKI. 185 Notably, SsD has been used in traditional Chinese medicine due to its anti-inflammatory and hepatoprotective properties, suggesting its potential safety. 5.2.2 | Other FTO inhibitors FTO inhibitor MO-I-500 has been reported to inhibit FTO demethylation in vitro, but its precise mode of action remains unclear due to the lack of crystal structure. 186O-I-500 significantly inhibits cell survival and colony formation of inflammatory breast cancer SUM149-MA cells compared to untreated cells or those treated with an inactive analogue, MO-I-100.187 However, resistance developed with prolonged co-culture in a glutaminefree medium, suggesting potential adaptive mechanisms or cellular changes overcoming the inhibitor's effects.Moreover, this inhibitory effect was not observed when cells were cultured in a medium containing glutamine, indicating that metabolic stress may play a role in MO-I-500's activity.
CS1 and CS2 are potent FTO inhibitors with undisclosed modes of action. 188Both induce apoptosis and G0 phase cell cycle arrest in human cells.In a patient-derived xeno-transplanted AML model, CS2 treatment reduces leukaemia infiltration and doubles survival.On the other hand, CS1 only shows enhanced anti-leukaemia activity when delivered in micelles. 188Furthermore, inhibition of FTO by CS1 or CS2 inhibits immune evasion in AML cells in vivo.More recently, Phan et al. demonstrated the in vivo anti-tumour efficacy of CS1 in CRC. 189

| FTO activators
Tricyclic antidepressants (TCAs) are among the first antidepressants developed. 190Imipramine (IMI) and Amitriptyline (AMI) activate FTO function and reduce m6A levels in N2a cells, 190 potentially contributing to their antidepressant effects.However, further research is needed to investigate their anti-tumour efficacy.

| ALKBH5 inhibitors
ALK-04 is a selective ALKBH5 inhibitor identified through in silico screening and the subsequent structure-activity relationship studies. 146Combining ALK-04 with immunotherapy significantly reduces tumour growth in mice, suggesting its potential to overcome anti-PD1 resistance and enhance immunotherapy effectiveness. 146V1035, a sodium channel blocker, reduced the migration and invasiveness of U87 glioblastoma cells. 191nterestingly, the reference sodium channel blocker TTX did not produce similar results, indicating that the antitumour effect is unrelated to sodium channel blocking.The study used SPILLO-PBSS software to explore the underlying mechanism and identified potential off-targets on a proteome-wide scale.The result indicated that MV1035 competitively binds to the cofactor site of ALKBH5, leading to increased cellular m6A-tagged mRNA and reduction of the oncoprotein CD73 expression. 191n virtual screening of 144,000 compounds from a library developed by the Institute for Molecular Medicine Finland identified 2-[(1-hydroxy-2-oxo-2-phenylethyl) sulfanyl]acetic acid and 4-{[(furan-2-yl)methyl]amino}-1,2-diazinane-3,6-dione.182 In vitro experiments on leukaemic and glioblastoma cells showed their potential as selective anti-proliferative agents for some cancer cell lines (i.e.HL-60, CCRF-CEM, and K562), but not for Jurkat or A172 cells.This highlights the complexity of m6A regulators' role in cancers, emphasising the need for further research to understand subtype-specific functions.182 Compound 20 m is a potent and selective ALKBH5 inhibitor, stabilising ALKBH5 in human hepatoma cells.However, its anti-proliferative effects in vitro or in vivo remain to be determined.192 Two compounds, Ena21 and Ena15, were discovered through the HTS of the Enamine Pharmacological Diversity Set.193 Docking studies revealed that Ena21 occupies the cofactor (2OG) binding site, suggesting it is a competitive inhibitor. Howver, Ena15 does not show such binding.Enzyme kinetic experiments support this conclusion.Inhibition of ALKBH5 with Ena21 and Ena15 successfully inhibits cell proliferation of glioblastoma multiforme-derived cells and decreases cell population in the synthesis phase of the cell cycle.the ChemBridge MicroFormat, the Unversity of Illinois Marvel library, and the NCI Diversity Set) identified BTYNB.194 BTYNB specifically targets and inhibits cell proliferation of IGF2BP1-positive cells but not IGF2BP1negative cells in melanoma and ovarian cancer cell lines.It also impairs cell proliferation and induces apoptosis in Neuroendocrine Neoplasm (NEN) cells.195 Testing on leukaemic cells showed decreased cell viability, increased cell death, and cell cycle arrest at S-phase.196 In vivo, BTYNB shows promising anti-tumour efficacy in xenograft models of intrahepatic cholangiocarcinoma and ovarian cancer.197,198 CuB, identified from HTS of 889 compounds from the Medicinal Natural Products Library, allosterically binds IGF2BP1, altering expression of downstream RNA such as c-MYC, Kras, and FSCN1.199 In vivo, CuB triggers apoptosis, recruits immune cells to the TME, and inhibits the expression of PD-L1.199

| Competitive inhibitor
Inhibitors of IGF2BP2 have also been identified through HTS of ~1200 compounds (Table 8).Ten compounds were identified, including 4 benzamidobenzoic acid class and 6 ureidothiophene class compounds, which inhibits cell proliferation in CRC cells. 200Three compounds tested show significant anti-tumour effects in a zebrafish xenograft model with minimal toxicity. 200However, poor membrane permeability limited their induction of cell death and a high dose is required.Virtual screening of 300,000 compounds via docking into the RNA-binding site of IGF2BP2 followed by cellular assay identified the inhibitor JX5. 138JX5 shows cytotoxicity against Jurkat cells with IGF2BP2 overexpressed but only mild inhibition in normal Jurkat cells, suggesting therapeutic potential for IGF2BP2-positive leukaemia.The anti-leukaemic effect was also confirmed in vivo.

| Other inhibitors
Another IGF2BP1 inhibitor identified via HTS is known as 7773. 201It binds a hydrophobic surface of IGF2BP1, inhibiting its binding to Kras RNA and other target RNAs.As a result, 7773 significantly reduces Kras expression and affects the downstream pathway, leading to a reduced pERK/ERK ratio.In vitro, 7773 inhibits cell migration in H1299, ES2, and HEK293 cell lines while cell proliferation remains unaffected.
To the best of our knowledge, no inhibitor of IGF2BP3 has been reported.However, Isoliquiritigenin, derived from the Chinese herb licorice, significantly reduces the expression of IGF2BP3.Downregulation of IGF2BP3 inhibits the downstream TWIST1 mRNA expression, consequently exhibiting an anti-tumour effect in NSCLC. 202

| LIMITATION OF TARGETING m6A REGULATORS
All the candidates discussed exhibit the potential to modulate m6A regulators, with many demonstrating potency and specificity as inhibitors.However, the inherent complexity of m6A modification poses a potential limitation for the application of these inhibitors.Much evidence suggests that targeting m6A regulators can be tumourspecific to some extent.An example is the overexpression of METTL3 in AML cells compared to healthy haematopoietic cells. 41Pharmacologically inhibiting METTL3 using STM2457 did not show adverse effects in the development of normal bone marrow cells. 153However, this remains to be seen in other cell types and in conditions whereby cells are exposed to stress or environments that can lead to adverse effects upon METTL3 depletion.
METTL3-mediated m6A methylation increases the maturation of miR-355, promoting stress granule (SG) NSCLC [202]  formation and reducing the apoptosis level of injured neurons and cells in acute ischemic stroke. 203Therefore, targeting m6A regulators may not be applicable to all patients, and the risk of causing stroke needs to be studied carefully.Moreover, knockdown of YTHDFs was also reported to reduce SG formation, 40 potentially leading to adverse effects if inhibited.However, another study argues that m6A modification only explains 6% of the variance in SG localisation and that it plays a minimal, if any, role in mRNA partitioning in SG formation.Nonetheless, evaluating the importance of a biological pathway by how often it occurs can be quite biased, while the existence of its complementary process is underappreciated.
Another major argument within the field includes the opposing role of the m6A regulators in certain cancers, examples include the conflicting results published around breast cancer.The reason for the observed inconsistency is underappreciated, which demands further research.This inconsistency is evident not only in the contrasting roles of m6A regulators across different and same cancer subtypes but also in their potential opposing impacts on cancer cells and immune cells.In the past decade, immunotherapy has brought about a revolutionary shift in the field of cancer treatment, where immune cells play a crucial, if not determinative, role in patient prognosis.Therefore, it becomes imperative to acknowledge the intricacies involved in specifically targeting m6A regulators within the appropriate immune cell types.
In the future, there are some strategies we can possibly use: (1) single-cell analysis of cell subsets: an in-depth exploration at the single-cell level is essential to unveil the specific functions of m6A regulators within different cell types and cancer types.This approach allows for a comprehensive understanding of the complex roles these regulators play in diverse cellular contexts; (2) development of efficient targeted delivery: advancements in biotechnology are necessary to enable the precise delivery of drugs; developing technologies that facilitate targeted delivery ensures that the impact of m6A modulation is concentrated in specific cell types, thus minimising unintended consequences, and enhancing therapeutic efficacy.(3) investigating the normal physiological role of m6A regulators: minimising side effects is crucial, especially when dealing with potentially adverse pathways associated with m6A mRNA modification.

DIRECTIONS
The complex roles of m6A regulators in cancer highlight the need for further research to unravel their subtypespecific functions.The diverse landscape of m6A regulators and their involvement in tumorigenesis underscore the importance of understanding their context-dependent roles in different cancer types.By investigating the subtype-specific functions of m6A regulators, we can uncover valuable insights that may guide precision cancer therapeutics.
We have discussed the current understanding of m6A regulators and their implications in cancer pathology.In this review, we focused on the potential of targeting these regulators as a therapeutic strategy, showcasing various inhibitors that have shown promise in preclinical studies.However, to fully harness the therapeutic potential of compounds targeting m6A regulators, it is crucial to delve into their efficacy in specific cancer subtypes, and consider the effects on immune cells and normal cells which could potentially influence cancer progression and lead to adverse effects.This precision medicine approach will enable the development of targeted therapies that address the specific molecular aberrations within individual tumours.

T A B L E 4
Examples of aberrant m6A regulators' expression in chemoresistance.

5
Examples of aberrant m6A regulators' expression in cancer immunology.
CS1 suppresses CRC cell proliferation in 6 colorectal cancer cell lines and in the 5-Fluorouracil resistant cell line.CS1 suppresses in vivo tumour growth in the HCT116 heterotopic model.CRC [188,189] Treatment leads to increased apoptosis and cell cycle arrest at the G0 phase in human AML cells.CS2 reduces leukaemia infiltration and doubled the overall survival in the patient-derived xenotransplantation AML model.AML MO-I-500 Inhibits survival and/or colony formation of SUM149-MA cells.

5. 4 |
M6A reader-IGF2BPs 5.4.1 | Allosteric inhibitor HTS of ~16,190 compounds from three libraries (i.e. B) Induced apoptosis of HCC cells.Exhibits anti-HCC effect through inducing apoptosis and recruiting immune cells to the tumour microenvironment as well as blocking PD-L1 expression. of leukaemic cells while increases cell death and cell cycle arrest at S-phase.Leukaemia Impairs viability of LUAD-derived A549 cells.Impairs the growth and spread of tumour cells with reduced tumour burden in ovarian cancer xenografted model.proliferation of HCT116, SW480, and Huh7 cells in 2D and 3D cultures.Inhibits tumour growth with minimal toxicity (zebrafish model). 55 Complex roles of aberrant m6A writers' expression in human cancers.Complex roles of aberrant m6A readers expression in human cancers.
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